Goat mGSCs Research Articles

Dmrt1 regulates the immune response by repressing the TLR4 signaling pathway in goat male germline stem cells.

Double sex and mab-3-related transcription factor 1 (Dmrt1), which is expressed in goat male germline stem cells (mGSCs) and Sertoli cells, is one of the most conserved transcription factors involved in sex determination. In this study, we highlighted the role of Dmrt1 in balancing the innate immune response in goat mGSCs. Dmrt1 recruited promyelocytic leukemia zinc finger (Plzf), also known as zinc finger and BTB domain-containing protein 16 (Zbtb16), to repress the Toll-like receptor 4 (TLR4)-dependent inflammatory signaling pathway and nuclear factor (NF)-κB. Knockdown of Dmrt1 in seminiferous tubules resulted in widespread degeneration of germ and somatic cells, while the expression of proinflammatory factors were significantly enhanced. We also demonstrated that Dmrt1 stimulated proliferation of mGSCs, but repressed apoptosis caused by the immune response. Thus, Dmrt1 is sufficient to reduce inflammation in the testes, thereby establishing the stability of spermatogenesis and the testicular microenvironment.

Interaction between DMRT1 and PLZF protein regulates self-renewal and proliferation in male germline stem cells

Double sex and mab-3 related transcription factor 1 (DMRT1) encodes a double sex/mab-3 (DM) domain, which is the most conserved structure that involved in sex determination both in vertebrates and invertebrates. This study revealed important roles of DMRT1 in maintaining self-renewal of male germline stem cells (mGSCs). Our results showed that insufficient expression of DMRT1 in mice testes resulted in decreased number of spermatogonial cells and collapse of testicular niche in vivo. Self-renewal and proliferation of mGSCs were inhibited. Based on the bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) assay, it was finally revealed that the interaction between DMRT1 and promyelocytic leukemia zinc finger (PLZF) protein was essential for maintaining self-renewal of mGSCs. Moreover, BTB domain of PLZF, DM and DMRT1 domain of DMRT1 were indispensable in mGSC, which were responsible for preserving the quantity of germ cells. Our research provided a new scientific basis for studying the mechanism of self-renewal and spermatogenesis in goat mGSCs.

P53 inhibits the proliferation of male germline stem cells from dairy goat cultured on poly‐L‐lysine

Male germline stem cells (mGSCs) can transmit genetic materials to the next generation and dedifferentiate into pluripotent stem cells. However, in livestock, mGSC lines are difficult to establish, because of the factors that affect their isolation and culture. The extracellular matrix serves as a substrate for attachment, and affects the fate of these stem cells. Poly-L-lysine (PL), an extracellular matrix of choice, inhibits and/or kills cancer cells, and promotes the attachment of stem cells in culture. However, how it affects the characteristics and potentials of these stem cells in culture, needs to be elucidated. Here, we isolated, enriched, and cultured dairy goat mGSCs on 5 types of extracellular matrices. To explore the best extracellular matrix to use for culturing them, the characteristics and proliferation ability of the cells were determined. Results showed that the cells shared several characteristics with previously reported mGSCs, including the poor effect of PL on their proliferative and colony-forming abilities. Further examination showed upregulation of p53 expression in these cells, which could be inhibiting their proliferation. When a p53 inhibitor was included in the culture medium, it was confirmed to be responsible for the inhibition of proliferation in mGSCs. Optimal concentration of the inhibitor in the culture of these cells was 5 µM. Furthermore, addition of the p53 inhibitor increased the expression of the markers of self-renewal and cell cycle in goat mGSCs. In summary, suppressing p53 is beneficial for the proliferation of dairy goat mGSCs, cultured on PL.

Functions of promyelocytic leukaemia zinc finger (Plzf) in male germline stem cell development and differentiation.

Promyelocytic leukaemia zinc finger (Plzf), also known as zinc finger and BTB domain containing 16 (ZBTB16) or zinc-finger protein 145 (ZFP145), is a critical zinc finger protein of male germline stem cells (mGSCs). Multiple lines of evidence indicate that Plzf has a central role in the development, differentiation and maintenance of many stem cells, including mGSCs, and Plzf has been validated as an essential transcription factor for mammalian testis development and spermatogenesis. This review summarises current literature focusing on the significance of Plzf in maintaining and regulating self-renewal and differentiation of mGSCs, especially goat mGSCs. The review summarises evidence of the specificity of Plzf expression in germ cell development stage, the known functions of Plzf and the microRNA-mediated mechanisms that control Plzf expression in mGSCs.

MiR-302 enhances the viability and stemness of male germline stem cells.

MicroRNAs were reported to be able to regulate mGSCs' self-renewal through post-transcriptional inhibition of gene expression. miR-302 worked as one important microRNA family existed mainly in human ESCs, and its role in mGSCs has not been reported yet. To elucidate the role of miR-302 in dairy goat mGSCs, the expression profile of miR-302 was explored through qPCR and FISH. Furthermore, to detect the function of miR-302, the expression vector containing miR-302 was transfected into mGSCs, and then, the cell cycle, the cell apoptosis and the genes associated with mGSCs' self-renewal and differentiation were examined. The results showed that miR-302 expressed in testis moderately and located on the basement of seminiferous tubes which shared the same location as mGSCs. Transfection of the vector containing miR-302 fragment into the immortalized mGSCs obviously enhanced the cell proliferation ability and the attachment ability, also, promoted the expression level of CD49f and OCT4. Also, miR-302 reduced the cell apoptosis and downregulated the expression of P21. miR-302 sustained mGSCs' proliferation in vitro.

The Modification of Tet1 in Male Germline Stem Cells and Interact with PCNA, HDAC1 to promote their Self-renewal and Proliferation.

Epigenetic modification plays key roles in spermatogenesis, especially DNA methylation dynamic is important in sustaining normal spermatogenesis. Ten-eleven translocation 1 (Tet1) is not only a key demethylase, which works in specific gene regions, but also crosstalks with partners to regulate epigenetic progress as protein complexes. Dairy goat is an important livestock in China, while the unstable culture system in vitro inhibits optimization of new dairy goat species. The study of epigenetic modification in male germline stem cells (mGSCs) is beneficial to the optimization of adult stem cell culture system in vitro, and the improvement of sperm quality and breeding of selected livestock. In our study, we not only analyzed the morphology, gene expression, DNA methylation and histone methylation dynamic in mouse Tet1 (mTet1) modified mGSCs, we also analyzed the stemness ability by in vivo transplantation and explored the functional mechanism of Tet1 in dairy goat mGSCs. The results showed mTet1 modified mGSCs had better self-renewal and proliferation ability than wild-type mGSCs, mTet1 could also up-regulate JMJD3 to decrease H3K27me3, which also showed to suppress the MEK-ERK pathway. Furthermore, Co-IP analysis demonstrated that TET1 interact with PCNA and HDAC1 by forming protein complexes to comprehensively regulate dairy goat mGSCs and spermatogenesis.

Modification of Tet1 and histone methylation dynamics in dairy goat male germline stem cells.

Tet (ten-eleven translocation) protein 1 is a key enzyme for DNA demethylation, which modulates DNA methylation and gene transcription. DNA methylation and histone methylation are critical elements in self-renewal of male germline stem cells (mGSCs) and spermatogenesis. mGSCs are the only type of adult stem cells able to achieve intergenerational transfer of genetic information, which is accomplished through differentiated sperm cells. However, numerous epigenetic obstacles including incomplete DNA methylation and histone methylation dynamics make establishment of stable livestock mGSC cell lines difficult. The present study was conducted to detect effects of DNA methylation and histone methylation dynamics in dairy goat mGSCs self-renewal and proliferation, through overexpression of Tet1. An immortalized dairy goat mGSC cell line bearing mouse Tet1 (mTet1) gene was screened and characteristics of the cells were assayed by quantitative real-time PCR (qRT-PCR), immunofluorescence assay, western blotting, fluorescence activated cell sorting (FACS) and use of the cell counting kit (CCK8) assay. The screened immortalized dairy goat mGSC cell line bearing mTet1, called mGSC-mTet1 cells was treated with optimal doxycycline (Dox) concentration to maintain Tet1 gene expression. mGSC-mTet1 cells proliferated at a significantly greater rate than wild-type mGSCs, and mGSCs-specific markers such as proliferating cell nuclear antigen (PCNA), cyclinD1 (CCND1), GDNF family receptor alpha 1 (Gfra1) and endogenic Tet1, Tet2 were upregulated. The cells exhibited not only reduction in level of histone methylation but also changes in nuclear location of that methylation marker. While H3K9me3 was uniformly distributed throughout the nucleus of mGSC-mTet1 cells, it was present in only particular locations in mGSCs. H3K27me3 was distributed surrounding the edges of nuclei of mGSC-mTet1 cells, while it was uniformly distributed throughout nuclei of mGSCs. Our results conclusively demonstrate that modification of mGSCs with mTet1 affected mGSC maintenance and seemed to promote establishment of stable goat mGSC cell lines. Taken together, our data suggest that Tet1 had novel and dynamic roles for regulating maintenance of pluripotency and proliferation of mGSCs by forming complexes with PCNA and histone methylation dynamics. This may provide new solutions for mGSCs stability and livestock mGSC cell line establishment.

CD49f promotes proliferation of male dairy goat germline stem cells.

CD49f enhances multipotency and maintains stemness in embryonic stem cells (ESCs), however, whether it would be effective in mGSCs has remained unclear. Moreover, better standards for mGSC enrichment and purification are necessary. The present study was conducted to determine roles of CD49f in mGSC enrichment and regulation. CD49f expression patterns were investigated in dairy goats. CD49f positive cells were purified and enriched using magnetic-activated cell sorting (MACS), and characteristics of the cultured cells were assayed using alkaline phosphatase (AP) analysis, quantitative real-time PCR (QRT-PCR) and immunofluorescence analysis. Furthermore, the exogenous CD49f gene was transfected into mGSCs and its effects were analysed. CD49f was found to be conserved in both mRNA and amino acid sequences and that it was an efficient marker for dairy goat mGSC identification, enrichment and purification. CD49f positive cells expressed higher levels of mGSC-specific markers, and proliferated faster than CD49f negative cells. Overexpression CD49f promoted proliferation of dairy goat mGSCs, and Oct4 expression was upregulated; histone H3-lysine 9 dimethylation (H3K9me2) was reduced. Taken together, our data suggest that CD49f plays novel and dynamic roles in regulating maintenance of pluripotency in mGSCs via Oct4 crosstalk and histone methylation dynamics,which may provide new solutions for mGSCs stability in vitro.

PLZF-Induced Upregulation of CXCR4 Promotes Dairy Goat Male Germline Stem Cell Proliferation by Targeting Mir146a.

Previous studies have shown that promyelocytic leukemia zinc finger (PLZF), chemokine (C-X-C motif) receptor 4 (CXCR4) and mir146a were associated with the self-renewal of mouse spermatogonial stem cells (SSCs); however, there is little information on their effects on the fate of livestock SSCs. Here, we have identified a regulatory pathway in dairy goat mGSCs, involving PLZF, mir146a and the SDF-1 receptor CXCR4. PLZF overexpression downregulated mir146a and simultaneously upregulated the expression of CXCR4 protein, whereas PLZF knockdown (siPLZF) induced the specifically opposite effects. The in vitro assays demonstrated that PLZF specifically interacts with and suppresses the mir146a promoter, and mir146a targets CXCR4 to impede its translation. The levels of ERK1/2 phosphorylation in the mGSCs overexpressed CXCR4 and PLZF were upregulated, respectively, whereas mir146a expression was decreased and CXCR4 protein was increased. Mir146a overexpression and siPLZF impaired mGSC proliferation and differentiation, however, Mir146a knockdown induced the opposite effects. The effects of PLZF and mir146a were mediated regulation by mir146a and CXCR4, respectively. Overexpression of CXCR4 or addition of CXCL12 in cultures of dairy goat mGSCs resulted in the upregulation of their signaling, and the phosphorylation of ERK1/2 was increased. Collectively, these findings indicate that PLZF is an important transcription factor in the regulation of the expression of CXCR4 to promote dairy goat mGSC proliferation by targeting mir146a.

Characterization of Immortalized Dairy Goat Male Germline Stem Cells (mGSCs)

Male germline stem cells (mGSCs), in charge for the fertility in male testis, are the only kind of adult stem cells that transmit genetic information to next generation, with promising prospects in germplasm resources preservation and optimization, and production of transgenic animals. Mouse male germline stem cell lines have been established and are valuable for studying the mechanisms of spermatogenesis. However, there is a lack of stable mGSC cell lines in livestock, which restricts the progress of transgenic research and related biotechnology. Here, we firstly established an immortalized dairy goat mGSC cell line to study the biological properties and the signaling pathways associated with mGSCs self-renewal and differentiation. The ectopic factors SV40 large T antigen and Bmi1 genes were transduced into dairy goat mGSCs, and the results showed that the proliferation of these cells that were named mGSCs-I-SB was improved significantly. They maintained the typical characteristics including the expression of mGSC markers, and the potential to differentiate into all three germ layers, sperm-like cells in vitro. Additionally, mGSCs-I-SB survived and differentiated into three germ layer cell types when they were transplanted into chicken embryos. Importantly, the cells also survived in mouse spermatogenesis deficiency model testis which seemed to be the golden standard to examine mGSCs. Conclusively, our results demonstrate that mGSCs-I-SB present the characteristics of mGSCs and may promote the future study on goat mGSCs.

Conservation and function of Dazl in promoting the meiosis of goat male germline stem cells

Dazl (deleted in azoospermia-like) is a conserved gene in mammalian meiosis, which encodes RNA binding protein required for spermatocyte meiosis. Up to date, the expression and function of Dazl in the goat testis are unknown. The objectives of this study were to investigate the expression pattern of Dazl in dairy goat testis and their function in male germline stem cells (mGSCs). The results first revealed that the expression level of Dazl in adult testes was significantly higher than younger and immature goats, and azoospermia and male intersex testis. The dairy goat Dazl is highly conserved analysed by several online and bioinformatics software, respectively. Over-expression of Dazl promoted the expression of meiosis-related genes in dairy goat mGSCs. The expression of Stra8 was up-regulated by over-expression of Dazl analysed by Luciferase reporter assay. Taken together, results suggest the Dazl plays an important role in dairy goat spermatogenesis and that over-expression of Dazl may promote Stra8 expression in dairy goat mGSCs.

Enrichment and characterization of Thy1-positive male germline stem cells (mGSCs) from dairy goat (Capra hircus) testis using magnetic microbeads

In mammalian testis, male germline stem cells (mGSCs) are originated from primordial germ cells and developed into spermatocyte and spermatid. In our previous studies, we had isolated a pluripotent mGSCs from goat testes and tested their pluripotency and differentiation potential in vitro and in vivo, which revealed that the isolated and cultured dairy goat mGSCs maintained the characteristics of mGSCs. However, Thy1, a marker of mGSCs, was not examined in detail. In this study, the dairy goat mGSCs were purified by differential plating followed by magnetic-activated cell sorting (MACS) using Thy1 antibody. The quantitative reverse transcription polymerase chain reaction and immunofluorescence analyses revealed that the transcription and expression of Thy1, CD49f, Plzf, Oct4, Gfra1, and Vasa were higher in Thy1-positive cells when compared with Thy1-negative cells. The detection results of culturing dairy goat mGSCs indicated that the Thy1-positive cells maintained the characteristics of mGSCs, grew relatively faster than Thy1-negative cells, and the percentage of alkaline phosphatase-positive cells and colonies were significantly higher in Thy1-positive mGSCs than Thy1-negative cells. Collectively, these results indicate that THY1 is a marker of undifferentiated spermatogonia in goat testes, the technique of magnetic-activated cell sorting using Thy1 antibody could be an efficient method to enrich mGSCs in goat.

GDNF Up-Regulates c-Myc Transcription via the PI3K/Akt Pathway to Promote Dairy Goat Male Germline Stem Cells (mGSC) Proliferation

Studies have demonstrated that regulation of GDNF on male germline stem cells (mGSCs) mainly through Ras/Erk1/2, Src family kinase and PI3K/Akt signaling pathways, but the signaling pathways GDNF-mediated are different when the species and cell lines varied. Whether GDNF regulates self-renewal of mGSCs isolated from livestock has not been reported. Here, we purified mGSCs from dairy goat testis using mixed enzymes and fibronectin. Immunofluoresce staining revealed the cultured dairy mGSCs expressed Vasa, Nanos2, Ngn3, Tert, Dazl, Lin28, Oct4, CD49f, Stra8 and GFRa1, reflecting that these cells were mGSCs phenotype. Then we cultured these dairy goat mGSCs in different concentrations of GDNF (0, 5, 10, or 20 ng mL−1) to optimize the best concentration of GDNF to sustain the dairy goat mGSCs self-renewal, after that the inhibitor of PI3K (LY294002, 10 µmol L−1) was added to the medium which contains the optimal concentration of GDNF we obtained by experiments. The mGSCs cultured in different media were compared through the population doubling time (PDT), capacity of cell proliferation evaluated by PCNA and BrdU immunofluorescence staining, RT-PCR, QRT-PCR, Western blotting and flow cytometry. Results showed that 10 ng mL−1 was the optimal concentration of GDNF to maintain goat mGSCs self-renewal and GDNF up-regulates c-Myc transcription via the PI3K/Akt pathway to promote goat mGSCs proliferation. This study provides us an efficient model to study the mechanism in mGSCs proliferation and differentiation in goat, and has important implications in unveiling signaling pathways in livestock GSCs.

The effects of Nanos2 on Boule and Stra8 in male germline stem cells (mGSCs)

The mitosis-meiosis switch is a key event in the differentiation of germ cells. Meiosis is important in development biology, however, it has not been clear what is the regulation mechanism in mammals. Our previous study showed that Boule could activate Stra8 directly and result in the meiosis initiation of dairy goat male germline stem cells (mGSCs). Nanos2, a RNA-binding protein, plays critical roles in the suppression of meiosis by preventing Stra8 expression and maintain the male germ cell development. The main purpose of this study was to explore whether Nanos2 represses Stra8 transcription through Boule or not. We found ectopic over-expression of Nanos2 in GC-1 and mGSCs down-regulated Stra8 transcription and translation, and Boule expression was not affected. It was in consistent with our expectation that RA could up-regulate Boule and Stra8 expression, but down-regulate Nanos2 expression in mGSCs. In dairy goat, the expression levels of Boule and Stra8 would rise with the increase of age, but the expression level of Nanos2 in 90 dpp and adult testis had not shown a clear change. In conclusion, Nanos2 represses Stra8 expression but not through Boule in dairy goat mGSCs.

Expression of miR‐34c in response to overexpression of Boule and Stra8 in dairy goat male germ line stem cells (mGSCs)

Reproduction is required for the survival of all mammalian animals. Spermatogenesis is an essential and complex developmental process that ultimately results in production of haploid spermatozoa. Recent studies demonstrated that Boule and stimulated by retinoic acid 8 (Stra8) played important roles in initiation meiosis in male germ cells. miR-34c is indispensable in the late steps of spermatogenesis; remarkably, the main function of miR-34c is to reduce cell proliferation potentiality and promote cellular apoptosis. The objectives of this study were to investigate the expression patterns of Boule, Stra8, P53 and miR-34c in dairy goat testis and their relationship in male germ line stem cells (mGSCs). The results first revealed the expression patterns of Boule, Stra8, P53 and miR-34c in 30 dpp, 90 dpp and adult testes of dairy goats. The expression levels of Boule, Stra8, P53 and miR-34c in adult dairy goat testes were significantly higher than that of 30 dpp. Overexpression of Boule and Stra8 promoted the expression of miR-34c in dairy goat mGSCs. In our previous study, we showed that miR-34c was P53 dependent in mGSCs. These results have shown that the up-regulation of miR-34c was not due to P53 protein activation but which might be caused by the up-regulation of Boule and Stra8 promoting the advance of meiosis. In addition, we found retinoic acid would decrease the expression of P53 and miR-34c, however, did not change the expression of c-Myc greatly. It suggested that the function of driving differentiation of dairy goat mGSCs by retinoic acid might not be caused by P53.

Expression pattern of Boule in dairy goat testis and its function in promoting the meiosis in male germline stem cells (mGSCs)

Boule is a conserved gene in meiosis, which encodes RNA binding protein required for spermatocyte meiosis. Deletion of Boule was found to block meiosis in spermatogenesis, which contributes to infertility. Up to date, the expression and function of Boule in the goat testis are not known. The objectives of this study were to investigate the expression pattern of Boule in dairy goat testis and their function in male germline stem cells (mGSCs). The results first revealed that the expression level of Boule in adult testes was significantly higher than younger and immature goats, and azoospermia and male intersex testis. Over-expression of Boule promoted the expression of meiosis-related genes in dairy goat mGSCs. The expression of Stra8 was up-regulated by over-expression of Boule analyzed by Western blotting and Luciferase reporter assay. While, Cdc25a, the downstream regulator of Boule, was found not to affect the expression of Stra8, and our data illustrated that Cdc25a did not regulate meiosis via Stra8. The expression of Stra8 and Boule was up-regulated by RA induction. Taken together, results suggest the Boule plays an important role in dairy goat spermatogenesis and that over-expression of Boule may promote spermatogenesis and meiosis in dairy goat.

Effect of GSK-3 inhibitor on the proliferation of multipotent male germ line stem cells (mGSCs) derived from goat testis

The glycogen synthase kinase 3 (GSK3) inhibitor, 6-bromoindirubin-3′-oxime (BIO), is a key regulator of many signaling pathways to maintain pluripotency of human and mouse embryonic stem cells (ESCs). However, the effect of BIO on derivation of dairy goat male germline stem cells (mGSCs) remains unclear. The objectives of this study were to investigate whether BIO influences derivation of dairy goat mGSCs. Dairy goat mGSCs were cultured in mTeSR containing BIO medium and its effects on the proliferation ability of goat mGSCs (derived from goats ≤2 mo of age) were evaluated by 5-Bromo-2-deoxyuridine (BrdU) incorporation and alkaline phosphatase (AP) staining. Furthermore, its effects on maintenance of the undifferentiated state of mGSCs in late passages of cultures, as well as the capacity of mGSCs to differentiate into embryoid bodies (EBs) were examined. The presence of BIO increased the mitosis index and the number of AP positive colonies, as well as expression of pluripotent markers, Oct4, Nanog, Sox2, C-myc, Klf4, E-cadherin, and the proliferative markers, Pcna and C-myc. In contrast, there was no significant change in expression of apoptosis markers, P53, P21 and cyclin-related genes (Cyclin A, CDK2, Cyclin D1), as determined by RT-PCR analysis. When mGSCs were cultured in mTeSR medium containing BIO, EBs were formed, which were capable of further differentiating into various cell types found in the three embryonic germ layers, as determined by immunofluorescence and/or histologic staining. In conclusion, adding BIO to cultures BIO significantly promoted establishment of goat mGSC colonies and maintained their undifferentiated state.

Pluripotent Male Germline Stem Cells from Goat Fetal Testis and Their Survival in Mouse Testis

Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatogenesis during their whole life. Studies have shown that mGSCs can be derived in vitro and resemble embryonic stem cells (ESCs) properties both in the mouse and humans. However, little is know about these cells in domestic animals. Here we report the first successful establishment of goat GSCs derived from 2-5-month fetal testis, and developmental potential assay of these cells both in vitro and in vivo. These cells express pluripotent markers such as Oct4, Sox2, C-myc, and Tert when cultured as human ESCs conditions. Embryoid bodies (EBs) formed by goat mGSCs were induced with 2 × 10(-6) M retinoic acid (RA). Immunofluorescence analysis showed that some cells inside of the EBs were positive for meiosis marker-SCP3, STRA8, and germ cell marker-VASA, and haploid marker-FE-J1, PRM1, indicating their germ cell lineage differentiation. Some cells become elongated sperm-like cells after induction. Approximately 34.88% (30/86) embryos showed cleavage and four embryos were cultured on murine fibroblast feeder and formed small embryonic stem like colonies. However, most stalled at four-cell stage after intracytoplasmic sperm injection (ICSI) of these cells. Transplantation of DAPI labeled mGSCs into the seminiferous tubules of busulfan-treated mice, and showed that mGSCs can colonize, self-renew, and differentiate into germ cells. Thus, we have established a goat GSC cell line and these cells could be differentiated into sperm-like cells in vivo and sperms in vitro, providing a promising platform for generation of transgenic goat for production of specific humanized proteins.